In this study, we evaluated the effect of oral administration of riboflavin combined with whole-body ultraviolet A (UVA) irradiation on the biochemical and biomechanical properties of sclera in a guinea pig model to control the progression of myopia. Experimental groups were administered 0.1% riboflavin solution with or without vitamin C by gavage from 3 days before myopic modeling and during the modeling process. Guinea pigs underwent 30 min of whole-body UVA irradiation after each gavage for 2 weeks. For control groups, guinea pigs were administered vitamin C and underwent either whole-body UVA irradiation without 0.1% riboflavin solution or whole-body fluorescent lamp irradiation with or without 0.1% riboflavin solution. Resultantly, myopia models were established with an increased axial length and myopic diopter. Compared with myopic eyes in the control groups, the net increase in axial length, diopter and strain assessment decreased significantly, and the net decrease in sclera thickness, ultimate load, and stress assessment decreased significantly in experimental groups. MMP-2 expression showed a lower net increase, while TIMP-2 expression showed a lower net decrease. In addition, hyperplasia of scleral fibroblasts was more active in myopic eyes of experimental groups. Overall, our results showed that oral administration of riboflavin with whole-body UVA irradiation could increase the strength and stiffness of sclera by altering the biochemical and biomechanical properties, and decreases in axial elongation and myopic diopter are greater in the guinea pig myopic model.